EP2083845B1 - Ablative immuntherapie - Google Patents

Ablative immuntherapie Download PDF

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Publication number
EP2083845B1
EP2083845B1 EP07874119.6A EP07874119A EP2083845B1 EP 2083845 B1 EP2083845 B1 EP 2083845B1 EP 07874119 A EP07874119 A EP 07874119A EP 2083845 B1 EP2083845 B1 EP 2083845B1
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cells
composition
tumor
allogeneic
patient
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French (fr)
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EP2083845A2 (de
EP2083845A4 (de
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Michael Har-Noy
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Immunovative Therapies Ltd
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Immunovative Therapies Ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/26—Universal/off- the- shelf cellular immunotherapy; Allogenic cells or means to avoid rejection
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/31—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterized by the route of administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates generally to immunotherapy and, more specifically, to therapeutic compositions for treating a tumor.
  • Harnessing the power of the immune system to treat chronic infectious diseases or cancer is a major goal of immunotherapy.
  • Active immunotherapy treatments are methods designed to activate the immune system to specifically recognize and destroy tumor or pathogen-infected cells.
  • active immunotherapy approaches have been used to prevent numerous infectious diseases, including small pox, rabies, typhoid, cholera, plague, measles, varicella, mumps, poliomyelitis, hepatitis B and the tetanus and diphtheria toxins.
  • Active immunotherapy concepts are now being applied to develop therapeutic cancer vaccines with the intention of treating existing tumors or preventing tumor recurrence as well as for treatment and prevention of chronic viral infection.
  • Many of these techniques have proven to successfully develop increased frequencies of immune cells in circulation that have the ability to specifically kill tumors or pathogen infected cells.
  • tumor escape mechanisms can overpower this immune response resulting in eventual tumor progression.
  • the innate immune response is able to slow down viral replication and activate cytokines which trigger the synthesis of antiviral proteins.
  • the adaptive immune system neutralizes virus particles and destroys infected cells.
  • viruses have developed a number of countermeasures to avoid immune attack and stay moving targets for the immune system.
  • the present invention relates to compositions for inducing a systemic, adaptive immune response against a tumor using a combination of an allogeneic cell therapy and subjecting the tumor to cellular distress, resulting in the liberation of tumor specific antigen(s).
  • the present invention includes a therapeutic composition according to claim 1.
  • the present invention includes a vaccine according to claim 10.
  • the present invention includes stimulating anti-tumor or anti-pathogen immunity in patients.
  • the stimulation involves first "priming" of the patient to develop Th1 anti-alloantigen immune memory by infusion of an aliquot of allogeneic cells. It is desired that the infusion of allogeneic cells stimulates the patient's immune system to react against the allogeneic cells. A time period is allowed to elapse until the patient's immune system is allowed to form an anti-allogeneic memory. In some embodiments, a patient may need a booster of allogeneic cells to develop the appropriate Th1 immune memory.
  • Th1 response refers to production of a cytokine profile that activates T-cells and macrophages. Th1 response is to be distinguished from Th2 response which activates mainly an immune response that depends upon antibodies and is antagonistic to the Th1 response.
  • the next step includes injury and/or death of cells within a tumor bed or pathogen-infected tissue after the patient develops sufficient anti-allogeneic Th1 immune memory.
  • Tissue injury or death releases cellular components and recruits scavenger cells to the injury site.
  • a variety of methods are known in the art to cause tissue injury or death within a tumor bed or pathogen infected tissue.
  • death is by necrosis, which causes recruitment of scavenger cells to the injury site.
  • tissue death or injury is by cryoablation or by irreversible electroporation. Alternatively, tissue is ablated ex-vivo and the released components injected into the patient.
  • the scavenger cells including immature dendritic cells can pick up antigens released from the damaged or dead tissues.
  • a second aliquot of allogeneic cells are injected intralesionally in order to cause the maturation of dendritic cells (DC) for the priming of Th1 immunity to the antigens.
  • intralesionally is meant administration of the composition of this invention through injection or otherwise directly into a cancerous area or tumor or a pathogen infected tissue.
  • all of the allogeneic cells administered to the patient are from the same source.
  • the allogeneic cells are administered between about 2 and about 24 hours after ablation of the tissue. This method is especially useful in the treatment of solid or metastatic tumors, particularly in patients with tumor lesions resident in the prostate, breast, bone, liver, lung, or kidney.
  • DTH delayed-type hypersensitivity
  • the general state of inflammation caused by the treatment process can serve to cause the DC to program T-cells to Th1 immunity against antigens in the damaged tissue resulting in a systemic adaptive immune response to the tumor or pathogen-infected cells and the disabling of tumor and pathogen-mediated immune avoidance mechanisms.
  • adaptive immunity is meant that the patient's defenses are mediated by B and T cells following exposure to antigen and that such defenses exhibit specificity, diversity, memory, and self/nonself recognition.
  • Such adaptive immunity is systemic within the patient.
  • Adaptive immunity is to be distinguished from innate immunity which is nonspecific and exists prior to exposure to the antigen.
  • ablation followed by administration of the allogeneic cells may be sufficient to generate the desired response.
  • the priming of the patient by a first aliquot of allogeneic cells may be omitted.
  • tissue from the tumor or infected by a pathogen is ablated followed by injection of an aliquot of allogeneic cells.
  • the present invention also includes vaccinating a patient having cancerous cells or an infected tissue.
  • the vaccination is, preferably, used for patients with hematological malignancies (e.g., Chronic Lymphocytic Leukemia, Multiple Myeloma, and non-Hodgkin's lymphomas) or viral infectious diseases (e.g., hepatitis B or C, herpes, HIV) and other disorders where the affected lesions are not easily assessable for ablation.
  • hematological malignancies e.g., Chronic Lymphocytic Leukemia, Multiple Myeloma, and non-Hodgkin's lymphomas
  • viral infectious diseases e.g., hepatitis B or C, herpes, HIV
  • the vaccination involves first "priming" of the patient to develop Th1 anti-alloantigen immune memory by infusion of a first aliquot of allogeneic cells. It is desired that the infusion of allogeneic cells stimulates the patient's immune system to react against the allogeneic cells. A time period is allowed to elapse until the patient's immune system is allowed to form an anti-allogeneic memory. In some embodiments, a patient may need a booster of allogeneic cells to develop the appropriate Th1 immune memory.
  • the next step includes injecting into the patient an antigenic composition that includes an autologous lysate containing antigens from the cancerous cells or the infected tissue.
  • This composition also includes an aliquot of the allogeneic cells, i.e. allogeneic cells that are from the same source as the allogeneic cells used in the priming step.
  • the injection of the antigenic composition can create a rejection response in the patient and can stimulate a delayed-type hypersensitivity response to the antigens.
  • the scavenger cells including immature dendritic cells can pick up the antigens from the autologous lysate.
  • the allogeneic cells can cause the maturation of dendritic cells for the priming of Th1 immunity to the antigens. It is desirable for the patient to develop a strong DTH reaction upon introduction of the allogeneic cells with the autologous lysate due to the fact that the patient has been primed to Th1 immunity against the allogeneic cells introduced by the first aliquot of allogeneic cells during the priming step.
  • the general state of inflammation caused by the treatment process can serve to cause the DC to program T-cells to Th1 immunity against the antigens in the autologous lysate resulting in a systemic adaptive immune response to the tumor or pathogen-infected cells and the disabling of tumor and pathogen-mediated immune avoidance mechanisms.
  • the present invention also provides for enhancing the immunogenicity of weakly immunogenic or non-immunogenic tumors and deviating an immune response from a non-protective immune response (e.g., Th2 response) to a protective immune response (e.g., Th1).
  • a non-protective immune response e.g., Th2 response
  • a protective immune response e.g., Th1
  • diseases include, for example, all types of cancers and diseases caused by infections with a variety of pathogens (e.g., Hepatitis viruses, fungal infections such as aspergillus, HIV, malaria, typhoid, cholera, herpes viruses, Chlamydia, and HPV).
  • pathogens e.g., Hepatitis viruses, fungal infections such as aspergillus, HIV, malaria, typhoid, cholera, herpes viruses, Chlamydia, and HPV.
  • the present invention also includes a therapeutic composition for treating a tumor or a pathogen in a patient.
  • the therapeutic composition preferably includes a priming composition and an antigenic composition.
  • the priming composition generally contains allogeneic cells which are injected into the patient to generate a rejection response by the patient's immune system in a manner that induces an allogeneic Th1 immunity.
  • the antigenic composition includes antigenic material from the tumor or pathogen-infected tissue and an aliquot of allogeneic cells.
  • the antigenic material is an autologous lysate containing antigens from the cancerous cells or from infected tissue.
  • the antigenic material can be derived from tissue necrosis of the tumor or the pathogen-infected tissue.
  • the antigenic material is derived from ablation of the tumor or pathogen-infected tissue. The ablation may be done in vivo or ex vivo.
  • the antigenic material includes heat shock proteins released upon ablation of the tissue from a tumor or pathogen-infected tissue.
  • the antigenic composition also includes allogeneic cells.
  • the antigenic material and the allogeneic cells may be combined together or packaged separately.
  • the antigenic composition including the antigenic material and the allogeneic cells when injected into the patient, can create a rejection response and stimulate a delayed-type hypersensitivity response to the antigens thereby acting as an adjuvant to the stimulation of systemic anti-tumor or anti-pathogen immunity in the patient.
  • the therapeutic compositions may include other components that act as adjuvants to the response generated by the priming composition and the antigenic composition.
  • the priming composition and antigenic composition may include other components generally found in therapeutic composition, for example, preservatives. The addition of these components are within the scope of this invention.
  • the therapeutic composition may only include the antigenic composition and not the priming composition.
  • the antigenic composition may be sufficient to obtain the desired immune response.
  • the present invention also includes a vaccine for a patient against a tumor or a pathogen.
  • the vaccine preferably includes a priming composition and an antigenic composition.
  • the priming composition generally contains allogeneic cells which are injected into the patient to generate a rejection response by the patient's immune system in a manner that induces an allogeneic Th1 immunity.
  • the antigenic composition includes antigenic material from the tumor or pathogen-infected tissue and an aliquot of allogeneic cells.
  • the antigenic material is an autologous lysate containing antigens from the cancerous cells or from infected tissue.
  • the antigenic material can be derived from tissue necrosis of the tumor or the pathogen-infected tissue.
  • the antigenic material is derived from ablation of the tumor or pathogen-infected tissue. The ablation may be done in vivo or ex vivo.
  • the antigenic material includes heat shock proteins released upon ablation of the tissue from a tumor or pathogen-infected tissue.
  • the antigenic composition also includes allogeneic cells.
  • the antigenic material and the allogeneic cells may be combined together or packaged separately.
  • the antigenic composition including the antigenic material and the allogeneic cells when injected into the patient, can create a rejection response and stimulate a delayed-type hypersensitivity response to the antigens thereby acting as an adjuvant to the stimulation of systemic anti-tumor or anti-pathogen immunity in the patient.
  • the vaccine may include other components that act as adjuvants to the response generated by the priming composition and the antigenic composition.
  • the priming composition and antigenic composition may include other components generally found in vaccines, for example, preservatives. The addition of these components are all within the scope of this invention.
  • the vaccine may only include the antigenic composition and not the priming composition.
  • the antigenic composition may be sufficient to obtain the desired immune response.
  • the therapeutic vaccines of the present invention are useful for the prevention and treatment of diseases such as cancer or chronic viral disease which develop and/or persist by suppressing or escaping the immune response.
  • the purpose of the priming step is to create anti-allogeneic Th1 immunity in a patient that can be recalled upon subsequent exposure to the alloantigens.
  • Priming occurs by exposing a patient to an aliquot of allogeneic cells and the subsequent rejection of these allogeneic cells when a second aliquot is administered to the patient by the patient's immune system resulting from immune memory.
  • the patients are not immunosuppressed prior to priming, as this will inhibit the ability of the patient to reject the infused allogeneic cells and will also inhibit the development of anti-alloantigen Th1 immunity.
  • the patient's immune system is skewed to generate Th1 immunity. It is preferable to manipulate the allogeneic cells such that Th1 and not Th2 immunity develops in response to the rejection of the allogeneic cells.
  • the patient's immune system can be skewed to produce Th-1 response by administering allogeneic cells that are producing Th1 cytokines (e.g., IFN-gamma and TNF-alpha) when infused. Th1 cytokines can assist in skewing the immune response to the alloantigens of Th1 type immunity. Other means of skewing a patient's immune system to produce Th-1 immunity can be employed.
  • the allogeneic cells used to first prime the patients and then later used for either intralesional administration (after induction of cell death) or as an adjuvant to a source of pathogenic or tumor material are preferably allogeneic activated T-cells, more preferably allogeneic activated CD4+ Th1 cells, more preferably allogeneic CD4+ T-cells that have differentiated into effector or memory cells and produce high levels of Type 1 cytokines, such as IL-2, IL-15, IFN-gamma, TNF-alpha and also express, preferably at high density, effector molecules such as CD40L, TRAIL and FasL on the cell surface but do not produce IL-4 or other Type 2 cytokines.
  • Type 1 cytokines such as IL-2, IL-15, IFN-gamma, TNF-alpha
  • effector molecules such as CD40L, TRAIL and FasL on the cell surface but do not produce IL-4 or other Type 2 cytokines.
  • CD40 ligation of innate immune cells has the capacity to induce high levels of the cytokine IL-12, which polarizes CD4+ T cells toward the Th1 type immunity, enhances proliferation of CD8+ T cells, and activates NK cells.
  • innate immune cells e.g., dendritic cells, macrophages and NK cells
  • cytokine IL-12 polarizes CD4+ T cells toward the Th1 type immunity, enhances proliferation of CD8+ T cells, and activates NK cells.
  • the activated allogeneic T-cells are administered to the patient, preferably intravenously, but can also be administered intradermally.
  • the allogeneic cells are preferably derived from a deliberately HLA-mismatched donor.
  • Preferred dosage in an aliquot of allogeneic cells for intravenous infusion is at least about 1 x 10 7 cells and more preferred is between about 1 x 10 8 to 1 x 10 10 cells. Dosages of allogeneic cells outside this range that can primarily generate an immune response are also within the scope of this invention.
  • Th1 anti-alloantigen immunity may take at least about 7 days.
  • the patient is allowed between about 7 days to about 14 days to develop Th1 anti-alloantigen immunity.
  • the development of Thi anti-alloantigen immunity can be measured by, for example, ELISPOT assay. Other methods of testing patients for development of Th1 anti-alloantigen immunity can be employed. If the Th1 anti-alloantigen immunity is weak, additional booster injections of allogeneic cells can be administered. Booster injections are preferably made intradermally to generate a delayed type hypersensitivity (DTH) reaction in the skin.
  • DTH delayed type hypersensitivity
  • allogeneic T-cells can be generated such that, upon, activation and infusion into a patient, a Th-1 immunity can be generated by the patient.
  • producing allogenic cells with the properties necessary for stimulation of anti-allogeneic Th1 immunity involves: (1) the collection of mononuclear cell source material by leukapheresis from normal screened donors; (2) the isolation of CD4 T-cells from the source material; (3) the activation of the CD4+ cells with immobilized anti-CD3 and anti-CD28 monoclonal antibodies (mAbs) on days 0, 3 and 6; (4) the activation of the cells again on day 9 with immobilized anti-CD3 and anti-CD28 mAbs and the infusion of the cells within 24h of activation.
  • mAbs monoclonal antibodies
  • necrosis includes a cell death by a variety of methods including cryoablation, irreversible electroporation, chemotherapy, radiation therapy, ultrasound therapy, ethanol chemoablation, microwave thermal ablation, radio frequency energy or a combination thereof.
  • cryoablation irreversible electroporation
  • chemotherapy radiation therapy
  • ultrasound therapy ultrasound therapy
  • ethanol chemoablation microwave thermal ablation
  • radio frequency energy radio frequency energy
  • the target tissue in order to cause death by necrosis, it is preferred that the target tissue is frozen.
  • Cryosurgery is a well-aimed and controlled procedure capable of inducing tissular necrosis by the application of liquid N2 or argon gas.
  • the biologic changes that occur during and after cryosurgery have been studied in vitro and in vivo. Tissue injury and necrosis is induced by cell freezing and by the vascular stasis that develops after thawing.
  • Cryosurgery in situ freezing
  • an antigenic stimulus capable of generating a specific immunologic response against autologous antigens of the frozen tissue.
  • Cryoablation can cause peptides to be released from lysed tumor or pathogen -infected cells for antigen processing by DC and creates a pro-inflammatory cytokine environment.
  • Cytokines released after cryoablation such as IL-1, IL-2, TNF- ⁇ , IFN- ⁇ , and GM-CSF can activate the T, NK, and Langerhans cells essential to an immune response capable of destroying cancer or pathogen infected cells.
  • the target tissue in order to cause death by necrosis, it is preferred that the target tissue is subject to irreversible electroporation.
  • Irreversible electroporation is a tissue ablation technique in which micro to milli-second electrical pulses are delivered to the tissue to produce cell necrosis through irreversible cell membrane permeabilization. In irreversible electroporation, the cellular membranes of the cells between the electrodes are disrupted causing cellular necrosis. Irreversible electroporation can cause antigens to be released from lysed tumor or pathogen -infected cells for antigen processing by DC and creates a pro-inflammatory cytokine environment.
  • a source of antigen can also be generated by isolating autologous chaperone proteins, also known as heat shock proteins (HSP), from dead infected tissue or tumors.
  • HSPs are among the major targets of the immune response to bacterial, fungal and parasitic pathogens.
  • Certain chaperones in extracellular milieu may also modulate innate and adaptive immunity due to their ability to chaperone polypeptides and to interact with the host's immune system, particularly professional antigen-presenting cells.
  • Vaccination with heat shock proteins from tumor have been shown to elicit an anti-tumor response.
  • Current studies indicate that the immunogenicity of HSPs is derived from the antigenic peptides with which they associate.
  • the purpose of the adjuvant step is to cause the maturation of DC to stimulate Th1 immunity against antigens taken up in the lesions containing dead target tissue.
  • This can be accomplished by the injection of the same allogeneic cells, i.e. allogeneic cells of the same origin as those used to prime the patient.
  • This aliquot of the allogeneic cells are, preferably, injected intralesionally, i.e. directly into the necrotic lesion caused by the cryoablation, or other cell death.
  • chaperone proteins are used as the source of antigen
  • the same allogeneic cells used to prime the patient are injected with the chaperone proteins, preferably intradermally.
  • the dosage of the allogeneic cells to generate the desired immune response is generally at least about 1 x 10 7 cells and more preferred is between about 1 x 10 8 to 1 x 10 10 cells. Dosages of allogeneic cells outside this range that can generate the desired immune response are also within the scope of this invention.
  • the preparation of the allogeneic cells is the same as described above.
  • the tissue resident DC termed immature DC
  • immature DC are able to capture Ag from the environment, but are deficient in stimulating T cells.
  • DC undergo a differentiation process called maturation, whereby they up-regulate the capacity to migrate to draining lymph nodes and present the captured antigens to T cells.
  • maturation a differentiation process
  • the DC has to integrate a number of maturation/differentiation stimuli.
  • exposure to pathogen or tumor-derived determinants, proinflammatory cytokines, and/or cell debris induces the first steps in the maturation process.
  • encounter of cognate CD4 + T cells provides additional differentiation stimuli to the DC, which regulate the survival of the activated T cells and the polarization of the CD4 + T cells.
  • the maturation of DC occurs at the site of antigen uptake and the recall rejection response serves as an adjuvant to provide the appropriate inflammatory danger signals necessary for DC maturation, migration to the lymph nodes and the programming for Th1 immunity against the antigens uptaken in the lesion.
  • mice were hosts and C57B1/6 (B6) mice were used as source of Th1 cells. All mice were 6 to 10 weeks old, were maintained in a specific pathogen-free facility at the Hadassah-Hebrew University Medical Center, and were treated on an approved animal protocol.
  • Spleen cells from male C57BL/6 mice were harvested and treated with ammonium chloride-potassium (ACK) buffer for lysis of red blood cells. Approximately 70-100 million cells were isolated per spleen. CD4+ T-cells were then purified by positive selection (purity >98%) using CD4 immunomagnetic particles on an MS column (Miltenyi Biotec, Germany), approximately 8-12 million CD4 cells were isolated with a yield of 50-60%. Th1 memory cells were generated by expansion with anti-CD3 and anti-CD28-coated paramagnetic beads (CD3/CD28 T-cell expander beads, Dynal/Invitrogen) at an initial bead:CD4 cell ratio of 3:1.
  • ACK ammonium chloride-potassium
  • the purified CD4 cells were incubated with 20 IU/mL recombinant mouse (rm)IL-2, 20 ng/mL rmIL-7, and 10 ng/mL rmIL-12 (Peprotech, New Jersey) and 10 ⁇ g/mL antimurine IL-4 mAb (Becton Dickenson) in RPMI 1640 media containing 10% FBS, penicillin-streptomycin-glutamine, nonessential amino acids (NEAA) (Biological Industries, Israel) and 3.3 mM N-acetyl-cysteine (NAC; Sigma) (complete media).
  • rm recombinant mouse
  • rmIL-7 20 IU/mL rmIL-7
  • 10 ng/mL rmIL-12 Peprotech, New Jersey
  • 10 ⁇ g/mL antimurine IL-4 mAb Becton Dickenson
  • RPMI 1640 media containing 10% FBS, penicillin-streptomycin-
  • cytokine-containing complete media with rmIL-2 and rmIL-7 was added to the CD4 cultures daily from days 3 to 6 to maintain the cell concentration between 0.5 and 1 x 10 6 cells/mL. Additional CD3/CD28 beads were added daily from day 3 to day 6. The number of beads added was calculated to maintain a 1:1 bead:cell ratio as the cells expanded. After 6 days in culture, the CD4 cells expanded approximately 80 to 100-fold and were harvested and debeaded by physical disruption and passage over a magnet. The phenotype of the harvested cells used in experiments were >95% CD4+, CD45RO+, CD62L lo , IFN- ⁇ + and IL-4-.
  • Biotinylated mouse anti-CD3 and anti-CD28 mAbs were each diluted in 400 ⁇ l of PBS to a final concentration of 25 ⁇ g/ml and then mixed in a 1:1 ratio so that the final volume was 800 ⁇ l.
  • 20 ⁇ l of Strepavidin-coated nanobeads (Miltenyi, Germany) were washed and diluted to a final volume of 200 ⁇ l in PBS.
  • the 800 ⁇ l of the CD3/CD28 mAb solution and the 200 ⁇ l of diluted nanobeads were then mixed so that the final concentration of each mAb was 10 ⁇ g/ml in a total volume of 1ml.
  • the mixture was placed on a rotating mixing device for 30 min at RT.
  • the mAb conjugated nanobeads were then passed over an MS column (Miltenyi, Germany) on a magnet and washed thoroughly. The retained nanobeads were then released from the column and resuspended in 200 ⁇ l of PBS. The nanobeads were not able to activate naive T-cells. Therefore, the nanobeads were tittered against harvested Th1 memory cells that had been previous activated 6 days prior with CD3/CD28 T-cell expander beads (Dynal, Norway). While there were slight variations per batch, generally 20 ⁇ l/10 7 cells was found to provide optimal activation of previously activated Th1 memory cells.
  • the harvested Th1 cells were incubated with a pre-tittered concentration of CD3/CD28-conjugated nanobeads prior to infusion.
  • the cells had to be incubated with the nanobeads for a minimum of 4h and a maximum of 18h.
  • Optimal activation caused production of IFN- ⁇ and upregulation of CD40L and FasL on the cell surface.
  • all infusions of CD3/CD28 cross-linked Th1 memory cells occurred after 4-8h of pre-incubation. Cells were thoroughly washed prior to infusion to remove any unassociated nanobeads.
  • Th1 memory cells used in these experiments expressed FasL and CD40L on the cell surface and produced in excess of 2000ng/ml/10 6 cells/6h IFN- ⁇ and less than 20pg/ml IL-4 per 10 6 cells/6h.
  • Th1 memory cells without CD3/CD28 cross-linking did not produce cytokines or express FasL or CD40L.
  • Cryotherapy was performed with a spherical nitrous oxide cryoprobe, 3 mm in diameter.
  • the gas was maintained at a pressure of 50 bars and the Joule-Thomson effect allowed to attain temperatures ranging from 30 to 40 °C in the tissue.
  • An incision was made in the centre of the tumor, the cryoprobe was placed in contact with the tumor (it was inserted 1-2 mm deep): the aim was to influence it by freezing but not to destroy it completely. Three cycles of rapid freezing (lasting for 20 s) followed by slow thawing were applied.
  • the ice ball was produced at the center of the lesion and reached about two thirds of the total tumor volume.
  • mice were treated intratumorally by injection of either: (a) saline; (b) saline+partial cryoablation of tumor; (c) allogeneic Th1 cells at a dose of 10 3 cells; or (d) allogeneic Th1 cells+partial cryoablation of tumor.
  • Th1 cells kill tumors by necrosis, which is thought to be a more pathological type of cell death than death by apoptosis (the type of death caused by chemotherapy). It is thought that the cryotherapy makes the tumors more immunogenic and therefore the combination of allogeneic Th1 cells with necrotic tumor death creates a type of tumor vaccine leading to systemic antitumor immunity.

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Claims (13)

  1. Therapeutische Zusammensetzung zur Behandlung eines Tumors bei einem Patienten, umfassend:
    eine antigene Zusammensetzung, die Tumorantigene umfasst, wobei die Tumorantigene Tumorgewebe sind, das Nekrose ausgesetzt ist, und ein Aliquot von allogenen aktivierten T-Zellen, wobei die antigene Zusammensetzung verabreichbar ist, um eine Immunantwort zu erzeugen, die als ein Adjuvans zur Aufnahme von Antigenen in der Zusammensetzung dient, wobei die anschließende Reifung der Antigen-präsentierenden Zellen des Patienten systemisch die Anti-Tumor-Immunität stimuliert.
  2. Zusammensetzung zur Verwendung nach Anspruch 1, wobei die Tumorantigene in der Zusammensetzung aus Nekrose des Tumorgewebes abgeleitet sind.
  3. Zusammensetzung zur Verwendung nach Anspruch 1, ferner umfassend eine Priming-Zusammensetzung, wobei die Priming-Zusammensetzung das Aliquot allogener Zellen umfasst.
  4. Zusammensetzung zur Verwendung nach Anspruch 3, wobei die allogenen Zellen der Priming-Zusammensetzung intravenös verabreicht werden.
  5. Zusammensetzung zur Verwendung nach Anspruch 3, wobei die allogenen Zellen der Priming-Zusammensetzung intradermal verabreicht werden.
  6. Zusammensetzung zur Verwendung nach Anspruch 1, wobei das Aliquot allogener Zellen zwischen etwa 1 x 108 und etwa 1 x 1010 Zellen umfasst.
  7. Zusammensetzung zur Verwendung nach Anspruch 1, wobei die allogenen Zellen Th1-Zytokine produzieren.
  8. Zusammensetzung zur Verwendung nach Anspruch 1, wobei dem Immunsystem des Patienten in zwischen etwa 7 Tagen bis etwa 14 Tagen ermöglicht wird, ein anti-allogenes Th1-Immungedächtnis zu bilden.
  9. Zusammensetzung zur Verwendung nach Anspruch 1, wobei die nach der Ablation injizierten allogenen Zellen intraläsional verabreicht werden.
  10. Impfstoff für einen Patienten gegen einen Tumor, umfassend:
    eine antigene Zusammensetzung, die antigenes Material aus dem Tumor umfasst, wobei die Tumorantigene Tumorgewebe sind, das Nekrose ausgesetzt ist, und ein Aliquot von allogenen aktivierten T-Zellen, wobei die antigene Zusammensetzung an den Patienten verabreichbar ist, um eine Abstoßungsreaktion zu erzeugen und eine Überempfindlichkeitsreaktion vom verzögertem Typ auf die Antigene zu stimulieren, wobei es damit als Adjuvant für die Stimulation der systemischen Antitumor-Immunität beim Patienten agiert.
  11. Impfstoff nach Anspruch 10 enthält ferner umfassend eine Priming-Zusammensetzung, wobei die Priming-Zusammensetzung das Aliquot allogener Zellen umfasst.
  12. Impfstoff nach Anspruch 10, wobei die Tumorantigene in der Zusammensetzung aus Nekrose des Tumors oder aus der Nekrose des mit Pathogen infizierten Gewebes abgeleitet sind.
  13. Impfstoff nach Anspruch 10, wobei das Aliquot allogener Zellen zwischen etwa 1 x 108 und etwa 1 x 1010 Zellen umfasst.
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